Microparticles as mediators and biomarkers of rheumatic disease.

Abstract

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and enter the blood to display pro-inflammatory and pro-thrombotic activities. MPs are 0.1-1.0 μm in size and incorporate nuclear, cytoplasmic and membrane molecules as they detach from cells. This process can occur with cell activation as well as cell death, with particles likely corresponding to blebs that form on the cell surface during apoptosis. To measure particle expression, flow cytometry allows determination of particle numbers based on size as well as surface markers that denote the cell of origin; platelet MPs are usually the most abundant type in blood. As shown in in vitro and in vivo systems, MPs can promote inflammation and thrombosis resulting from their content of cytokines like IL-1 and pro-coagulant molecules like tissue factor. Certain particle types can be anti-inflammatory, however, suggesting a range of immunomodulatory activities depending on the cell of origin. Studies on patients with a wide range of rheumatic disease show increased MP numbers in blood, with platelet and endothelial particles associated with vascular manifestations; increased numbers of particles also occur in the joint fluid where they may drive cytokine production and activate synoviocytes. In autoimmune diseases such as SLE and RA, MPs may also contribute to disease pathogenesis by the formation of immune complexes. MPs thus represent novel subcellular structures that can impact on the pathogenesis of rheumatic disease and serve as biomarkers of underlying cellular disturbances.

Mechanism of MP release by nucleated cells. The figure illustrates the two main mechanisms for the generation and release of MPs from nucleated cells and differences in particle composition. During activation of cells, blebs (shown as small circles) form at the cell surface and undergo release; since the cell is otherwise intact, the released MPs do not contain nuclear components. In contrast, during apoptosis, the cell undergoes drastic changes, including shrinkage, nuclear fragmentation and migration of nuclear constituents into blebs that form at the cell surface. MP release usually occurs during the later stages of apoptosis. As such, MPs can contain nuclear components that are shown in the darker blue. MPs differ from apoptotic bodies that are the collapsed remains of apoptotic cells.

Size distribution of MPs by flow cytometry. Jurkat cells were treated with 1 μM staurosporine for 18 h to induce apoptosis. The culture supernatant was analysed by flow cytometry by side scatter (SSC), as depicted in the filled profile. The size of the MPs and cells (indicated in the filled peaks) was determined by reference to fluorescent microspheres (Fluorospheres, Size Kit #2, Invitrogen, Carlsbad, CA, USA) of 0.1, 0.5, 1.0 and 2.0 μm sizes (dotted peaks). The first filled peak, comprising the MP fraction, is indicated by the horizontal gate. The second filled peak beyond the horizontal gate comprises the Jurkat cells. The FACS instrument was calibrated using diluent alone to ensure that no background events were detected by side scatter.

Total and IgG-bound MPs in the plasma of SLE patients. MPs were isolated from 21 SLE patient (○) and normal control (•) plasmas by differential centrifugation and analysed by FACS by side scatter and binding of a phycoerythrin-labelled goat anti-human IgG antibody. (A) Total MP numbers determined by side scatter. (B) The number of particles with IgG. The distribution of MP counts is indicated by box plots. The grey box indicates samples within the range of the 25th and 75th percentiles. The broken horizontal line indicates the population means and the solid line indicates the population median values. Although the total number of MPs in SLE plasmas did not differ significantly from those of normal controls (*), the numbers of IgG-positive MPs in SLE patient plasmas was significantly higher compared with 12 normal controls (†P = 0.00038). Adapted from [], with permission from Elsevier.